The temporal profile of activity-dependent presynaptic phospho-signalling reveals long lasting patterns of post-stimulus regulation

ABSTRACT: Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the post-stimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. CaMKIIα responded rapidly, scaled with stimulus strength and had long-lasting activity. MAPK/ERK was the main protein kinase predicted to control a distinct and significant pattern of post-stimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain, Cell Culture

SUBMITTER: Mark Graham  

LAB HEAD: Mark E. Graham

PROVIDER: PXD010007 | Pride | 2019-01-28

REPOSITORIES: Pride